The first triple-helical structure of nucleic acids was discovered more than 30 years ago (Felsenfeld, G., et al. (1957) J. Am. Chem. Soc. 79:2023-2024). While the biological roles of such structures are still open to question, their chemical characteristics have been considerably elucidated in recent works (for review, see Wells, R. D., et al. (1988) FASEB J. 2:2939-2949). The most well-characterized triplex is the one formed between a double-stranded homopurine-homopyrimidine helix and a single-stranded homopyrimidine tract. In this type of triple-helix, the third homopyrimidine strand binds to the major groove, parallel to the homopurine strand of Watson-Crick double-helical DNA, via Hoogsteen hydrogen bonding. The third-strand thymine (T) recognizes adenine-thymine (AT) base pairs forming T-A-T triplets, and the third-strand cytosine (C), protonated at its N-3 position, recognizes guanine-cytosine (G.multidot.C) base pairs forming C.sup.+ .multidot.G.multidot.C triplets.
Homopyrimidine oligonucleotides have been shown to form local triplexes with corresponding homopurine sites in larger double-stranded DNAs. Such oligonucleotide-directed triplex formation has been successfully applied in the recent development of sequence-specific artificial rare-cutting endonucleases (Moser, H. E. & Dervan, P. B. (1987) Science 238:645-650; Le Doan, T., et al. (1987) Nucleic Acids Res. 15:7749-7760), in which oligonucleotides and equipped metal chelates or photoactive groups function as DNA binding and cleaving "domains," respectively. Also, single-site enzymatic cleavage of the yeast genome was achieved by the triplex-mediated "Achilles' heel cleavage" procedure (Strobel, S. A. & Dervan, P. B. (1991) Nature (London) 350:172-174), in which a triplex-forming oligonucleotide, instead of a DNA binding protein (Koob, M. and Szybalski, W. (1990) Science 250:271-273), was used to protect the targeted single-site from DNA methyltransferase. Such triplex-mediated DNA cleavage techniques provide valuable tools for genome analysis. Specific inhibition of DNA-binding proteins (e.g., transcription factors or replication factors) by triplex formation (Maher, L. J., III, et al. (1989) Science 245:725-730; Francois, J.-C., et al. (1989) Biochemistry 28:9617-9619; Hanvey, J. C., et al. (1990) Nucleic Acids Res. 18:157-161) may provide a principle for the development of antiviral or anticancer drugs. Based on the stability of such triplexes during gel electrophoresis (Lyamichev, V. I. et al. (1988) Nucleic Acids Res. 16:2165-2178), a unique procedure for labeling specific DNA fragments was also devised to facilitate restriction mapping of cosmid inserts (Moores, J. C. (1990) Strategies 3:23-24, 29).
Typically, specific DNA is isolated from heterogeneous DNA mixtures using conventional hybridization based methods (e.g., colony or plaque hybridization (Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor, Cold Spring Harbor Press)). These well-established methods while quite reliable have some practical drawbacks. First, they require time-consuming and labor-intensive steps of filter preparation that often limit the number of clones that can be screened. Furthermore, since these procedures include prior denaturation steps and other treatments that destroy the integrity of the target DNA molecules, one has to reisolate the corresponding clones from the original plates to obtain intact DNA molecules for further biological biochemical manipulations. Third, sequences toxic to the host sometimes hamper successful cloning. Fourth, the natural modifications of the target DNA are not maintained during cloning. Finally, despite recent development of Yeast Artificial Chromosome (YAC) vectors, it is still difficult to clone very large DNAs. Obviously, non-cloning-based biochemical methods to isolate specific DNA from a complex mixture would be of some help with these problems. However, such methods are still not satisfactory. Biochemical purification by density and size fractionation after cleavage with restriction enzymes can be applied only in limited instances (Tsujimoto, Y. and Suzuki, Y. (1984) Proc. Natl. Acad. Sci. USA 81:1644-1648). The polymerase chain reaction (PCR) fulfills some of these needs and provides large amounts of DNAs (Mullis, K. B. and Falocna, F. A. (1987) Methods Enzymol 155:335-350), but it is currently limited to relatively short (&lt;10 kb) DNA fragments. Furthermore, natural modifications of the original DNA cannot be maintained. Although an effective method of affinity chromatography for DNAs was reported (Tsurui, H. et al. (1990) Gene 88:233-239), it requires the prior denaturation of target DNA molecules and elution by denaturation.
Several screening procedures, potentially applicable to large DNAs, that keep the target DNA in its native double stranded form have been developed using RecA protein (Honigberg, S. M., et al. (1986) Proc. Natl. Acad. Sci. USA 83:9586-9590; Rigas, B., et al. (1986) Proc. Natl. Acad. Sci. USA 83:9591-9595). An affinity capture procedure using hybridization at the end of a large DNA fragment was also reported (Kandpal, R., et al. (1990) Nucleic Acids Res. 18:1789-1795). However, these procedures include the handling of DNAs in solution, in at least several steps, which inevitably breaks large DNAs into smaller pieces.
The present invention provides a new rapid method for isolating sequence specific intact double stranded DNA from a heterogeneous mix in a sample by forming an intermolecular triplex with the target DNA and separating the triplex from the mix by means of a solid phase. It also provides a new method for isolating very large sequence specific intact double stranded DNAs by capturing them during electrophoresis on a solid phase embedded in the electrophoretic gel.